臺灣博碩士論文加值系統

犬小病毒為一種具有高度傳染的疾病，外型呈二十面體對稱且小型無封套病毒，為5.2kb單股DNA，傳染途徑由犬隻直接或間接接觸受感染的犬隻的糞便。此病毒是由felie panleukopaenia virus（FPLV）經由少量病毒外殼基因序列突變而來;CPV可分為兩種血清型CPV1和CPV2，其中以CPV2對犬隻最為嚴重, CPV2導致犬出血性腸炎有時犬心肌炎，狗舔舐攜帶CPV2的糞便、土壤和污染物而染病，該病毒伴隨吞咽在喉的淋巴組織中複製，之後迅速的進入血液循環，這病毒能攻擊正在分裂的細胞特別是那些在淋巴結和腸隱窩以及骨髓。我們從已感染的狗篩選出CPV2病毒並藉由PCR方式增殖VP2片段，隨後將此VP-2片段裝載入pET32a以原核表現系統表現並藉由Ni2+純化管柱純化蛋白，接著利用抗His及感染過CPV隻狗血進行西方點墨法和ELISA證實VP2蛋白抗原性，之後免疫老鼠以生產單株抗體，以及免疫紐西蘭大白兔以生產多株抗體，已經篩選出有效對抗CPV-VP2的單株抗體共有13株以及多株抗體，經由實驗測試篩選之後，挑選出其中一株編號CPV-mAb-6單株抗體野外株具有良好的結合力，再利用三明治免疫吸附法利用單株抗體15 µg/ml的濃度及多株抗體1.5 µg/ml的濃度偵測野外株檢體，野外株檢體共有14隻，經由聚合酶連鎖反應測試之後，其中陰性對照組為2隻，陽性對照組為12隻，三明治免疫吸附法測試結果與經由聚合酶連鎖反應測試相符，所以經由三明治免疫吸附法實驗證實我們所生產的單株抗體及多株抗體可以100%檢測犬隻是否有得病，在未來上希望可以增加犬隻的數量，進而增加可性度以及靈敏度，希望將此藉以預防疾病並輔助注射疫苗前篩選。

Canine parvovirus (CPV) is a small and non-enveloped icosohedral symmetry virus, containing a 5.2kb single-stranded DNA. The virus is from feline panleukopaenia virus (FPLV) throµgh a small number of mutations in the single capsid protein. There are two serotypes of Canine Parvovirus. The type 2 of canine parvovirus (CPV-2) causes hemorrhagic enteritis and sometimes myocarditis in dogs. Dogs are infected through oral contact with CPV2 in feces, infected soil, or fomites that carry the virus, following by ingestion of the feces, hence, the virus replicates in the lymphoid tissue in the throat, and then spread to the bloodstream. From there, the virus attacks rapidly dividing cells, notably those in the lymph nodes, intestinal crypts, and within bone marrow. We isolated the CPV from infected dogs and amplified the VP2 fragments by PCR. And then, the VP2 gene of CPV was constructed into the E. coil expression vector and purified by Ni2+ affinity column. The antigenicity of recombinant VP2 protein was characterized by Western blot with Anti-HIS antibody and CPV infected dog serum. The Anti-CPV-VP2 was screened 13 monoclonal antibody and polyclonal antibody . single out of which a number Anti-CPV monoclonal antibody-6 field strains with excellent adhesive force. The Anti-CPV polyclonal antibodies were coated on the ELISA plate and 10 dogs’ faecal specimens were used as the antigene. Then the captured CPV were probed with Anti- CPV monoclonal antibody-6 in sandwhich ELISA. Then the captured CPV were probed with Anti-CPV monoclonal antibody in sandwhich ELISA. The faecal samples were used for CPV-VP2 gene detection by PCR. demonstrates that polyclonal antisera gave visually positive reactions with 12 out of 14samples. we need more wild type sample to test The strip of evaluate the specificity and Sensitivity. The were generated and used for strip development for pre-vaccination screening and disease prevention.

目錄
中文摘要 I
Abstract III
謝 誌 V
圖表目錄 X
第一章 緒言 1
第二章 文獻探討 4
2.1犬小病毒歷史背景 4
2.2犬小病毒的宿主範圍 5
2.3犬小病毒的起源 5
2.4犬小病毒的結構及特性 5
2.4.1基因結構 5
2.4.2蛋白結構與特性 6
2.4.3犬小病毒的基因型 6
2.5犬小病毒傳染途徑及致病機轉 7
2.5.1臨床傳染途徑 7
2.5.2細胞感染途徑 7
2.6犬小病毒的致病機轉 8
2.6.1腸胃炎 8
2.6.2心肌炎 9
2.6.3 免疫抑制型 9
2.7犬小病毒的臨床病徵 9
2.7.1腸胃炎 9
2.7.2心肌炎 10
2.7.3全身性感染 10
2.8犬小病毒的病理學變化 10
2.8.1腸胃炎 10
2.8.2心肌炎 11
2.8.3全身性感染 11
2.9犬小病毒的診斷方法 11
2.9.1血清學診斷 11
2.9.2病毒鑑定 12
2.9.3電子顯微鏡 12
2.9.4血球凝集試驗 12
2.9.5酵素連結免疫吸附法 12
2.9.6聚合酵素連鎖反應 13
2.9.7試紙條 13
2.10免疫層析試紙檢測 14
2.10.1免疫層析試紙檢測歷史 14
2.10.2免疫層析試紙檢測原理 14
2.10.3膠體金 15
2.10.4三明治型檢測模式 15
2.11研究目的與動機 16
第三章 材料與方法 17
3.1實驗方法與架構 17
3.2.病毒的診斷 18
3.2.1 檢體的來源 18
3.2.2檢體的採集與保存 18
3.2.3病毒DNA的萃取 18
3.3CPV之VP2全長基因增幅 19
3.3.1CPV之VP2基因引子設計 19
3.3.2聚合酵素連鎖反應 19
3.3.3 DNA電泳分析 20
3.3.4 PCR產物回收 20
3.4將目標基因選殖到保存載體中 21
3.4.1接合選殖載體 21
3.4.2 DH5a勝認細胞的製備 21
3.4.3轉型作用 21
3.4.4藍白篩選 22
3.4.5萃取DNA質體與純化 22
3.4.6 細菌的保存 23
3.5核酸之定序與分析 23
3 6基因重組原核表現系統 23
3.6.1將選殖載體的產物裝載在pET32a載體並確認 23
3.6.2 BL21 Codon plas勝任細胞的配置 23
3.6.3蛋白質誘導 24
3.6.4蛋白質萃取 24
3.6.5蛋白質純化 24
3.7檢測有無目標蛋白及抗原性 25
3.7.1 SDS-PAGE蛋白電泳 25
3.7.2西方轉漬分析與冷光呈色 26
3.7.3 ELISA分析步驟 27
3.8單株抗體製作 28
3.8.1骨髓瘤細胞培養 28
3.8.2免疫BALB/c小鼠Pet32a-CPV-VP2 蛋白 28
3.8.3細胞融合反應 28
3.8.4酵素免疫分析法偵測融合細胞 29
3.8.5極限稀釋 30
3.8.6利用西方墨點法偵測融合細胞是否有產生抗體 30
3.9利用小鼠大量生產單株抗體 31
3.9.1融合細胞免疫BALB/c產生大量的抗體 31
3.9.2融合細胞上清液純化抗體 32
3.9.3純化BALB/c的CPV mAb 32
3.10三明治免疫分析法 32
3.11用pET32a-CPV-VP2 蛋白免疫紐西蘭大白兔產生多株抗體 33
3.12免疫層析試紙檢測 34
第四章 結果 35
4.1利用PCR方式檢測野外犬隻檢體 35
4.2確認CPV-VP2片段並裝載到yT&;A vector 35
4.3確認CPV-VP2片段並裝載到pET32a vector 35
4.4IPTG誘導蛋白SDS-page結果 35
4.5確認pET32a-CPV-VP2蛋白藉由西方墨點法分析 36
4.6 pET32a-CPV-VP2蛋白純化以SDS-page分析結果 36
4.7 pET32a-CPV-VP2蛋白純化以西方墨點法分析 36
4.8 測定蛋白濃度 36
4.9利用ELISA 分析偵測pET32a-CPV-VP2的靈敏度 37
4.10利用ELISA 分析偵測pET32a-CPV-VP2對血清的靈敏度 37
4.11融合瘤細胞的融合及極限稀釋結果 37
4.12極限稀釋後利用ELISA確定結果 37
4.13單株抗體免疫BLAB/c小鼠產生腹水之西方墨點法確定結果 37
4.14純化腹水IgG抗體SDS-page結果 38
4.15三明治免疫競爭分析法 38
4.16重組蛋白免疫兔子產生血清之SDS-PAGE 38
4.17純化兔子血清抗體西方墨點法確定結果 39
4.18三明治型ELISA單抗稀釋倍數 39
4.19三明治型ELISA 39
4.20試紙條測試 40
4.21台灣野外株CPV親源樹分析 40
第五章 討論 62
第六章 參考文獻 68
作者簡介 80


圖表目錄
Fig. 1 Detection of CPV DNA by PCR in feces sample. 41
Fig. 2 Analyses of CPV -VP2 gene by restriction enzymes (EcoRI and Sal I) on yT&;A clone. 42
Fig. 3 Analyses of the CPV VP2 gene by restriction enzymes (EcoRI and Sal I) digestion of pET32-a clons. 43
Fig. 4 The recombinant CPV-VP2 protein were analyzed by SDS-PAGE and Western blot. 44
Fig. 5 The nicklion column purified CPV-VP2 were analysed wish SDS-pege. 45
Fig. 6 The recombinant CPV-VP2 protein were analyzed by Western blot 46
Fig. 7 Establishment of the coating concentration of VP2 protein in ELISA experiment. 47
Fig. 8 Establishment the properety dilution of canine serum in ELISA experiment. 48
Fig. 9 The hybridoma cells morphology. 49
Fig. 10 The monoclonal antibodies specific reacted with CPV-VP2 protein. 50
Fig. 11 The CPV-VP2 protein can be recognized by monoclonal antibodies. 51
Fig. 12 Analysis of the purified monoclonal antibody was analyzed with SDS-PAGE. 52
Fig. 13 Study of the binding epitope property of different monoclonal antibodies with competitive ELISA. 53
Fig. 14 The purified polyclonal antibody was analyzed with SDS-PAGE. 54
Fig. 15 Analysis of antigenicity of the purificated rabbit polyclonal antibody with western. 55
Fig. 16 Establishment of the concentration of monocloned antibody in ELISA experiment. 56
Fig . 17 The specificity of homemade CPV detection sandwhich ELISA. 57
Fig . 18 Detectionm of the sensitivity of Anti-CPV IC test strip. 58
Fig .19 Phylogenetic analysis of different canine parvovirus based on deduced amino acid sequence of CPV-VP2 gene. 60

Table. 1 CPV-VP2 gene information used in this study. 59
Table. 2 The percentage identities and diversities of deduced nucleotide and amino acid sequences of the CPV-VP2 gene among CPV-VP2 strains in this study. 61

參考文獻
1. 王恆雄. 臺中地區所發生之犬出血性胃腸炎臨床病例之研究. 中華民國獸醫學會雜誌 6:107-112, 1980.

2. 朱瑞民、馮翰鵬、劉振軒. 犬傳染性出血性腸炎. In: 動物醫學1981, p. 11:36.

3. 梁朝雄. 犬病毒性出血性胃腸炎病例. 動物醫學 6:11-13, 1979.

4. 陳威達. 台中地區犬小病毒感染情形及 VP1/VP2 重組蛋白之表現. 2003.

5. Appel MJ, Cooper BJ, Greisen H, Scott F, and Carmichael LE. Canine viral enteritis. I. Status report on corona- and parvo-like viral enteritides. Cornell Vet 69: 123-133, 1979a.

6. Appel MJ, Scott FW, and Carmichael LE. Isolation and immunisation studies of a canine parco-like virus from dogs with haemorrhagic enteritis. Vet Rec 105: 156-159, 1979b.

7. Atwell RB, and Kelly WR. Canine parvovirus: a cause of chronic myocardial fibrosis and adolescent congestive heart failure. J Small Anim Pract 21: 609-620, 1980.

8. Basak S, and Turner H. Infectious entry pathway for canine parvovirus. Virology 186: 368-376, 1992.

9. Basak S, Turner H, and Parr S. Identification of a 40- to 42-kDa attachment polypeptide for canine parvovirus in A72 cells. Virology 205: 7-16, 1994.

10. Binn LN, Lazar EC, Eddy GA, and Kajima M. Recovery and characterization of a minute virus of canines. Infect Immun 1: 503-508, 1970.

11. Birnbaum S, Uden C, Magnusson CG, and Nilsson S. Latex-based thin-layer immunoaffinity chromatography for quantitation of protein analytes. Analytical biochemistry 206: 168-171, 1992.

12. Black JW, Holscher MA, Powell HS, and Byerly CS. Parvoviral enteritis and panleukopenia in dogs. Vet Med Small Anim Clin 74: 47-50, 1979.

13. Buonavoglia C, Martella V, Pratelli A, Tempesta M, Cavalli A, Buonavoglia D, Bozzo G, Elia G, Decaro N, and Carmichael L. Evidence for evolution of canine parvovirus type 2 in Italy. J Gen Virol 82: 3021-3025, 2001a.

14. Burtonboy G, Coignoul F, Delferriere N, and Pastoret PP. Canine hemorrhagic enteritis: detection of viral particles by electron microscopy. Arch Virol 61: 1-11, 1979.

15. Carlson J, Rushlow K, Maxwell I, Maxwell F, Winston S, and Hahn W. Cloning and sequence of DNA encoding structural proteins of the autonomous parvovirus feline panleukopenia virus. J Virol 55: 574-582, 1985.

16. Carman PS, and Povey RC. Experimental challenge of dogs with canine parvovirus-2. Vet Rec 107: 447, 1980.

17. Carmichael LE, and Binn LN. New enteric viruses in the dog. Adv Vet Sci Comp Med 25: 1-37, 1981a.

18. Carmichael LE, Joubert JC, and Pollock RV. Hemagglutination by canine parvovirus: serologic studies and diagnostic applications. Am J Vet Res 41: 784-791, 1980.

19. Carmichael LE, Schlafer DH, and Hashimoto A. Pathogenicity of minute virus of canines (MVC) for the canine fetus. Cornell Vet 81: 151-171, 1991.

20. Chandler J, Robinson N, and Whiting K. Handling false signals in gold-based rapid tests. IVD Technology 7: 34-45, 2001.

21. Clinton GM, and Hayashi M. The parvovirus MVM: a comparison of heavy and light particle infectivity and their density conversion in vitro. Virology 74: 57-63, 1976.

22. Decaro N, Martella V, Desario C, Bellacicco AL, Camero M, Manna L, d'Aloja D, and Buonavoglia C. First detection of canine parvovirus type 2c in pups with haemorrhagic enteritis in Spain. J Vet Med B Infect Dis Vet Public Health 53: 468-472, 2006.

23. Dykman LA, and Bogatyrev VA. Use of the dot-immunogold assay for the rapid diagnosis of acute enteric infections. FEMS immunology and medical microbiology 27: 135-137, 2000.

24. Eugster AK, Bendele RA, and Jones LP. Parvovirus infection in dogs. J Am Vet Med Assoc 173: 1340-1341, 1978.

25. Gao S, Nie C, Wang J, Kang L, Zhou Y, and Wang JL. Colloidal gold-based immunochromatographic test strip for rapid detection of abrin in food samples. Journal of food protection 75: 112-117, 2012.

26. Glickman LT, and Appel MJ. Parvovirus infection and distemper vaccination. J Am Vet Med Assoc 178: 1029-1031, 1981.

27. Greenwood N, Chalmers W, Baxendale W, and Thompson H. Comparison of isolates of canine parvovirus by monoclonal antibody and restriction enzyme analysis. Veterinary Record 138: 495-496, 1996.

28. Hirayama K, Kano R, Hosokawa-Kanai T, Tuchiya K, Tsuyama S, Nakamura Y, Sasaki Y, and Hasegawa A. VP2 gene of a canine parvovirus isolate from stool of a puppy. J Vet Med Sci 67: 139-143, 2005.

29. Hueffer K, and Parrish CR. Parvovirus host range, cell tropism and evolution. Curr Opin Microbiol 6: 392-398, 2003.

30. Huxtable CR, Howell JM, Robinson WR, Wilcox GE, and Pass DA. Sudden death in puppies associated with a suspected viral myocarditis. Aust Vet J 55: 37-38, 1979.

31. Koptopoulos G, Papadopoulos O, Papanastasopoulou M, and Cornwell HJ. Presence of antibody cross-reacting with canine parvovirus in the sera of dogs from Greece. Vet Rec 118: 332-333, 1986.

32. Krakowka S, Olsen RG, Axthelm MK, Rice J, and Winters K. Canine parvovirus infection potentiates canine distemper encephalitis attributable to modified live-virus vaccine. J Am Vet Med Assoc 180: 137-139, 1982.

33. Langeveld JP, Casal JI, Cortes E, van de Wetering G, Boshuizen RS, Schaaper WM, Dalsgaard K, and Meloen RH. Effective induction of neutralizing antibodies with the amino terminus of VP2 of canine parvovirus as a synthetic peptide. Vaccine 12: 1473-1480, 1994.

34. Langeveld JP, Casal JI, Vela C, Dalsgaard K, Smale SH, Puijk WC, and Meloen RH. B-cell epitopes of canine parvovirus: distribution on the primary structure and exposure on the viral surface. J Virol 67: 765-772, 1993.
35. Lenghaus C, and Studdert MJ. Generalized parvovirus disease in neonatal pups. J Am Vet Med Assoc 181: 41-45, 1982.

36. Lenghaus C, Studdert MJ, and Finnie JW. Acute and chronic canine parvovirus myocarditis following intrauterine inoculation. Aust Vet J 56: 465-468, 1980.

37. Lin LR, Fu ZG, Dan B, Jing GJ, Tong ML, Chen DT, Yu Y, Zhang CG, Yang TC, and Zhang ZY. Development of a colloidal gold-immunochromatography assay to detect immunoglobulin G antibodies to Treponema pallidum with TPN17 and TPN47. Diagnostic microbiology and infectious disease 68: 193-200, 2010.

38. Lopez de Turiso JA, Cortes E, Ranz A, Garcia J, Sanz A, Vela C, and Casal JI. Fine mapping of canine parvovirus B cell epitopes. J Gen Virol 72 ( Pt 10): 2445-2456, 1991.

39. Lopez de Turiso JAL, Cortes E, Martinez C, de Ybanez RR, Simarro I, Vela C, and Casal I. Recombinant vaccine for canine parvovirus in dogs. Journal of virology 66: 2748-2753, 1992.

40. Macartney L, McCandlish IA, Thompson H, and Cornwell HJ. Canine parvovirus enteritis 1: Clinical, haematological and pathological features of experimental infection. Vet Rec 115: 201-210, 1984a.
41. Macartney L, McCandlish IA, Thompson H, and Cornwell HJ. Canine parvovirus
enteritis 2: Pathogenesis. Vet Rec 115: 453-460, 1984b.

42. Macartney L, Parrish CR, Binn LN, and Carmichael LE. Characterization of minute virus of canines (MVC) and its pathogenicity for pups. Cornell Vet 78: 131-145, 1988.

43. Macintire DK, and Smith-Carr. S. Canine parvovirus . Part II. clinical signs, diagnosis, and treatment. Small Animal 19(13): 291-302., 1997.

44. Marsh M, and Helenius A. Virus entry into animal cells. Adv Virus Res 36: 107-151, 1989.

45. Martyn JC, Davidson BE, and Studdert MJ. Nucleotide sequence of feline panleukopenia virus: comparison with canine parvovirus identifies host-specific differences. J Gen Virol 71 ( Pt 11): 2747-2753, 1990.

46. McCandlish IA, Thompson H, Cornwell HJ, Laird H, and Wright NG. Isolation of a parvovirus from dogs in Britain. Vet Rec 105: 167-168, 1979.

47. Mildbrand MM, Teramoto YA, Collins JK, Mathys A, and Winston S. Rapid detection of canine parvovirus in feces using monoclonal antibodies and enzyme-linked immunosorbent assay. Am J Vet Res 45: 2281-2284, 1984.

48. Mochizuki M, Harasawa R, and Nakatani H. Antigenic and genomic variabilities among recently prevalent parvoviruses of canine and feline origin in Japan. Veterinary microbiology 38: 1-10, 1993a.

49. Mochizuki M, Horiuchi M, Hiragi H, San Gabriel MC, Yasuda N, and Uno T. Isolation of canine parvovirus from a cat manifesting clinical signs of feline panleukopenia. J Clin Microbiol 34: 2101-2105, 1996.

50. Mochizuki M, San Gabriel MC, Nakatani H, Yoshida M, and Harasawa R. Comparison of polymerase chain reaction with virus isolation and haemagglutination assays for the detection of canine parvoviruses in faecal specimens. Res Vet Sci 55: 60-63, 1993b.

51. Nakamura M, Tohya Y, Miyazawa T, Mochizuki M, Phung HT, Nguyen NH, Huynh LM, Nguyen LT, Nguyen PN, Nguyen PV, Nguyen NP, and Akashi H. A novel antigenic variant of Canine parvovirus from a Vietnamese dog. Arch Virol 149: 2261-2269, 2004.

52. Oh JS, Ha GW, Cho YS, Kim MJ, An DJ, Hwang KK, Lim YK, Park BK, Kang B, and Song DS. One-step immunochromatography assay kit for detecting antibodies to canine parvovirus. Clin Vaccine Immunol 13: 520-524, 2006.

53. Paek SH, Lee SH, Cho JH, and Kim YS. Development of rapid one-step immunochromatographic assay. Methods 22: 53-60, 2000.

54. Paradiso PR, Rhode SL, 3rd, and Singer, II. Canine parvovirus: a biochemical and ultrastructural characterization. J Gen Virol 62 (Pt 1): 113-125, 1982.

55. Park JS, Choi BK, Vijayachandran LS, Ayyappan V, Chong CK, Lee KS, Kim SC, and Choi CW. Immunodetection of Canine Parvovirus (CPV) in clinical samples by polyclonal antisera against CPV-VP2 protein expressed in Esherichia coli as an antigen. J Virol Methods 146: 281-287, 2007.

56. Parker J, and Parrish CR. Canine parvovirus host range is determined by the specific conformation of an additional region of the capsid. Journal of virology 71: 9214-9222, 1997.

57. Parrish CR. Emergence, natural history, and variation of canine, mink, and feline parvoviruses. Adv Virus Res 38: 403-450, 1990.

58. Parrish CR, Aquadro CF, Strassheim ML, Evermann JF, Sgro JY, and Mohammed HO. Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus. J Virol 65: 6544-6552, 1991.

59. Parrish CR, Have P, Foreyt WJ, Evermann JF, Senda M, and Carmichael LE. The global spread and replacement of canine parvovirus strains. J Gen Virol 69 ( Pt 5): 1111-1116, 1988.

60. Pletcher JM, Toft JD, 2nd, Frey RM, and Casey HW. Histopathologic evidence for parvovirus infection in dogs. J Am Vet Med Assoc 175: 825-828, 1979.

61. Reed AP, Jones EV, and Miller TJ. Nucleotide sequence and genome organization of canine parvovirus. J Virol 62: 266-276, 1988.

62. Rimmelzwaan GF, Carlson J, UytdeHaag FG, and Osterhaus AD. A synthetic peptide derived from the amino acid sequence of canine parvovirus structural proteins which defines a B cell epitope and elicits antiviral antibody in BALB c mice. J Gen Virol 71 ( Pt 11): 2741-2745, 1990.

63. Robinson WF, Huxtable CR, Pass DA, and Howell JM. Clinical and electrocardiographic findings in suspected viral myocarditis of pups. Aust Vet J 55: 351-355, 1979.
64. Robinson WF, Wilcox GE, and Flower RL. Canine parvoviral disease: experimental reproduction of the enteric form with a parvovirus isolated from a case of myocarditis. Vet Pathol 17: 589-599, 1980.

65. Sagazio P, Tempesta M, Buonavoglia D, Cirone F, and Buonavoglia C. Antigenic characterization of canine parvovirus strains isolated in Italy. Journal of virological methods 73: 197-200, 1998.

66. Schunck B, Kraft W, and Truyen U. A simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces. J Virol Methods 55: 427-433, 1995.

67. Senda M, Parrish CR, Harasawa R, Gamoh K, Muramatsu M, Hirayama N, and Itoh O. Detection by PCR of wild-type canine parvovirus which contaminates dog vaccines. J Clin Microbiol 33: 110-113, 1995.

68. Shyu RH, Shyu HF, Liu HW, and Tang SS. Colloidal gold-based immunochromatographic assay for detection of ricin. Toxicon : official journal of the International Society on Toxinology 40: 255-258, 2002.

69. Steinel A, Parrish CR, Bloom ME, and Truyen U. Parvovirus infections in wild carnivores. J Wildl Dis 37: 594-607, 2001.

70. Steinel A, Venter E, Van Vuuren M, Parrish C, and Truyen U. Antigenic and genetic analysis of canine parvoviruses in southern Africa. The Onderstepoort journal of veterinary research 65: 239, 1998.

71. Suikkanen S, Saajarvi K, Hirsimaki J, Valilehto O, Reunanen H, Vihinen-Ranta M, and Vuento M. Role of recycling endosomes and lysosomes in dynein-dependent entry of canine parvovirus. J Virol 76: 4401-4411, 2002.

72. Surleraux M, Bodeus M, and Burtonboy G. Study of canine parvovirus polypeptides by immunoblot analysis. Arch Virol 95: 271-281, 1987.

73. Surleraux M, and Burtonboy G. Structural polypeptides of a canine parvovirus: study by immunoadsorption and sequential analysis of infected cells. Brief report. Arch Virol 82: 233-240, 1984.

74. Thomas NJ, Foreyt WJ, Evermann JF, Windberg LA, and Knowlton FF. Seroprevalence of canine parvovirus in wild coyotes from Texas, Utah, and Idaho (1972 to 1983). J Am Vet Med Assoc 185: 1283-1287, 1984.

75. Truyen U. Emergence and recent evolution of canine parvovirus. Vet Microbiol 69: 47-50, 1999.

76. Truyen U. Evolution of canine parvovirus--a need for new vaccines? Vet Microbiol 117: 9-13, 2006.

77. Truyen U, Evermann JF, Vieler E, and Parrish CR. Evolution of canine parvovirus involved loss and gain of feline host range. Virology 215: 186-189, 1996.

78. Vihinen-Ranta M, Kalela A, Makinen P, Kakkola L, Marjomaki V, and Vuento M. Intracellular route of canine parvovirus entry. J Virol 72: 802-806, 1998.

79. Wilsmore AJ, and Davidson I. 'Clearview' rapid test compared with other methods to diagnose chlamydial infection. Vet Rec 128: 503-504, 1991.

80. Zarnke RL, and Ballard WB. Serologic survey for selected microbial pathogens of wolves in Alaska, 1975-1982. J Wildl Dis 23: 77-85, 1987.

81. Albanell J, Codony J, Rovira A, Mellado B, and Gascón P. Mechanism of action of anti-HER2 monoclonal antibodies: scientific update on trastuzumab and 2C4. New Trends in Cancer for the 21 st Century 253-268, 2003.

82. Buonavoglia C, Martella V, Pratelli A, Tempesta M, Cavalli A, Buonavoglia D, Bozzo G, Elia G, Decaro N, and Carmichael L. Evidence for evolution of canine parvovirus type 2 in Italy. Journal of General Virology 82: 3021, 2001b.

83. Buonavoglia D, Cavalli A, Pratelli A, Martella V, Greco G, Tempesta M, and Buonavoglia C. Antigenic analysis of canine parvovirus strains isolated in Italy. The new microbiologica 23:
93, 2000.
84. Carmichael L, and Binn L. New enteric viruses in the dog. Advances in veterinary science and comparative medicine 25: 1, 1981b.

85. Carstens CP, and Waesche A. Codon bias-adjusted BL21 derivatives for protein expression. Homo 11: 11.13, 1999.

86. De Ybanez R, Vela C, Cortes E, Simarro I, and Casal J. Identification of types of canine parvovirus circulating in Spain. The Veterinary record 136: 174, 1995.

87. Decaro N, and Buonavoglia C. Canine parvovirus–a review of epidemiological and diagnostic aspects, with emphasis on type 2c. Veterinary microbiology 2011.

88. Marty M, Cognetti F, Maraninchi D, Snyder R, Mauriac L, Tubiana-Hulin M, Chan S, Grimes D, Antón A, and Lluch A. Randomized phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2–positive metastatic breast cancer administered as first-line treatment: The M77001 study group. Journal of clinical oncology 23: 4265-4274, 2005.